The invention relates to the construction and use of multigene expression vectors useful to enhance production of materials by multienzyme pathways. In particular, the construction and use of multigene vectors encoding proteins in the polyhydroxyalkanoate biosynthetic pathway is disclosed.
Metabolic engineering is a process by which the normal metabolism of an organism is altered to change the concentration of normal metabolites, or to create novel metabolites. This process often involves introduction or alteration of numerous enzymatic steps, and thus often requires introduction of multiple genes. An efficient system for introducing and expressing multiple genes is therefore desirable. In prokaryotes such as Escherichia coli, introduction of multiple genes is relatively straightforward in that operons can be constructed to express multiple open reading frames, or multiple complete genes can be expressed from a single plasmid. However, introduction of pathways into plants is more difficult due in part to the complexity of plant genes, the difficulty of constructing vectors harboring multiple genes for expression in plants, and the difficulty of introducing large vectors intact into plants.
Polyhydroxyalkanoates are bacterial polyesters that accumulate in a wide variety of bacteria. These polymers have properties ranging from stiff and brittle plastics to rubber-like materials, and are biodegradable. Because of these properties, polyhydroxyalkanoates are an attractive source of non-polluting plastics and elastomers.
Currently, there are approximately a dozen biodegradable plastics in commercial use that possess properties suitable for producing a number of specialty and commodity products (Lindsay, Modern Plastics 2: 62, 1992). One such biodegradable plastic in the polyhydroxyalkanoate (PHA) family that is commercially important is Biopol(trademark), a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This bioplastic is used to produce biodegradable molded material (e.g., bottles), films, coatings, and in drug release applications. Biopol(trademark) is produced via a fermentation process employing the bacterium Ralstonia eutropha (Byrom, D. Trends Biotechnol. 5: 246-250, 1987). (R. eutropha was formerly designated Alcaligenes eutrophus [Yabuuchi et al., Microbiol. Immunol. 39:897-904, 1995]). The current market price is $6-7/lb, and the annual production is 1,000 tons. By best estimates, this price can be reduced only about 2-fold via fermentation (Poirier, Y. et al., Bio/Technology 13: 142, 1995). Competitive synthetic plastics such as polypropylene and polyethylene cost about 35-45¢/lb (Layman, Chem. and Eng News, p. 10 (Oct. 31, 1994). The annual global demand for polyethylene alone is about 37 million metric tons (Poirier, Y. et al., Int. J. Biol. Macromol. 17: 7-12, 1995). It is therefore likely that the cost of producing P(31HB-co-3HV) by microbial fermentation will restrict its use to low-volume specialty applications.
Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids (Anderson. A. J. and Dawes, E. A. Microbiol. Rev. 54: 450-472, 1990; Steinbxc3xcchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, New York), pp. 123-213, 1991); Poirier, Y., Nawrath, C. and Somerville, C. Bio/Technology 13: 143-150, 1995). Polyhydroxybutyrate (PHB) is the most well-characterized PHA. High molecular weight PHB is found as intracellular inclusions in a wide variety of bacteria (Steinbxc3xcchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D.: (MacMillan, New York), pp. 123-213, 1991). In Ralstonia eutropha, PHB typically accumulates to 80% dry weight with inclusions being typically 0.2-1 xcexcm in diameter. Small quantity of PHB oligomers of approximately 150 monomer units are also found associated with membranes of bacteria and eukaryotes, where they form channels permeable to calcium (Reusch, R. N., Can. J. Microbiol. 41 (Suppl. 1): 50-54, 1995). High molecular weight polyhydroxyalkanoates have the properties of thermoplastics and elastomers. Numerous bacteria and fungi can hydrolyze polyhydroxyalkanoates to monomers and oligomers, which are metabolized as a carbon source. Polyhydroxyalkanoates have accordingly attracted attention as a potential source of renewable arid biodegradable plastics and elastomers. PHB is a highly crystalline polymer with rather poor physical properties, being relatively stiff and brittle (de Koning, G., Can. J. Microbiol. 41 (Suppl. 1): 303-309, 1995). In contrast, PHA copolymers containing monomer units ranging from 3 to 5 carbons for short-chain-length PHA (SCL-PHA), or 6 to 1,4 carbons for medium-chain-length PHA (MCL-PHA), are less crystalline and more flexible polymers (de Koning, G., Can. J. Microbiol. 41 (Suppl. 1): 303-309, 1995).
PHB has been produced in the plant Arabidopsis thaliana expressing the R. eutropha PHB biosynthetic enzymes (Poirier, Y. et al., Science 256: 520-523, 1992; Nawrath, C., et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764, 1994). In plants expressing the. PHB pathway in the plastids, leaves accumulated up to 14% PHB per gram dry weight (Nawrath, C., et al., Proc. Natl. Acad Sci. U.S.A. 91: 12760-12764, 1994). High-level synthesis of PHB in plants opened the possibility of utilizing agricultural crops as a suitable system for the production of polyhydroxyalkanoates on a large scale and at low cost (Poirier, Y. et al., Bio/Technology 13: 143-150, 1995; Poirier, Y. et al., FEMS Microbiol. Rev. 103: 237-246, 1992; Nawrath, C., et al. Molecular Breeding 1: 105-22, 1995). PHB was also shown to be synthesized in insect cells expressing a mutant fatty acid synthase (Williams, M. D., et al., Appl. Environ. Microbiol. 62: 2540-2546, 1996), and in yeast expressing the R. eutropha PHB synthase (Leaf, T. A., et al. Microbiol. 142: 1169-1180, 1996).
A number of pseudomonads, including Pseudomonas putida and Pseudomonas aeruginosa, accumulate MCL-PHAs when cells are grown on alkanoic acids (Anderson, A. J. and Dawes, E. A. Microbiol. Rev. 54: 450-472, 1990; Steinbxc3xcchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, New York), pp. 123-213, 1991; Poirier, Y., Nawrath, C. and Somerville, C. Bio/Technology 13: 143-150, 1995). The nature of the PHA produced is related to the substrate used for growth and is typically composed of monomers which are 2n carbons shorter than the substrate. These studies indicate that MCL-PHAs are synthesized by the PHA synthase from 3-hydroxyacyl-CoA intermediates generated by the xcex2-oxidation of alkanoic acids (Huijberts, G. N. M., et al. Appl. Environ. Microbiol. 58: 536-544, 1992; Huijberts, G. N. M., et al., J. Bacteriol. 176: 1661-1666, 1994).
Chen et al. (Nature Biotech., 16: 1060-1064, 1998; reviewed by Gelvin, S. B., Nature Biotech., 16: 1009-1010, 1998) describes the cobombardment of embryogenic rice tissues with a mixture of 14 different pUC based plasmids. Integration of multiple transgenes was observed to occur at one or two genetic loci.
Creating a transgenic host cell or plant that produces multiple enzymes within a biosynthetic pathway is often a daunting task. Individual vectors must be created for each enzyme. Transformation of the host cell or plant is typically accomplished by one of three general methods: serial transformation, parallel transformation followed by crossing, or batch transformation. Each method has serious practical drawbacks.
Serial transformation involves transforming a host cell or plant with the first vector, selecting and characterizing the transformed cell or plant, transforming with the second vector, and so on. This process can become quite laborious and time consuming.
Parallel transformation followed by crossing involves separately transforming cells with each of the individual vectors, and subsequently mating or crossbreeding the transformed cells or plants to obtain a final cell or plant which contains all of the individual sequences. This is a lengthy process, especially for the crossbreeding of plant lines.
Batch transformation involves a single transformation event involving all of the individual vectors. A wide array of cells are produced, each containing between none and all of the vectors. While only a single transformation is required, extensive characterization of the resulting cells is necessary. As the number of vectors increases, it is increasingly likely that no cells will be obtained containing all of the vectors. If no desired transformed cells are identified, the transformation must be repeated.
An additional concern with all three of these methods is that they do not allow any control over the relative copy numbers of the individual vectors in the transformed cell or plant. It would be desirable to have a transformation method that permits control of the relative copy numbers of the individual sequences in the transformed cell or plant, and also coordinates the positional effect of the insertion locus.
There exists a need for improved materials and methods for the preparation of transgenic organisms transformed with multiple nucleic acid sequences encoding members of a multi-enzyme biosynthetic pathway.
The invention involves the construction and use of nucleic acid segments and vectors containing multiple sequences encoding members of a biosynthetic pathway. The resulting vector allows a single transformation event to produce a transformed cell or plant containing all of the nucleic acid sequences. Furthermore, the researcher has total control over the number of copies of each coding sequence within the constructed vector. Single or multiple copies of each coding sequence may easily be designed into the vector.
An unexpected beneficial result of the invention is that organisms transformed with a multi-enzyme coding vector produce the biosynthetic product in higher yield than organisms produced by serial transformation, parallel transformation with crossing, or batch transformation methods.
The invention is directed generally towards the construction and use of nucleic acid segments comprising sequences encoding multiple enzymes in a multi-enzyme biosynthetic pathway. The biosynthetic pathway may generally be any biosynthetic pathway. Examples of such multi-enzyme biosynthetic pathways are the TCA cycle, polyketide synthesis pathway, carotenoid synthesis, glycolysis, gluconeogenesis, starch synthesis, lignins and related compounds, production of small molecules that serve as pesticides, fungicides, or antibiotics, and polymer synthesis pathways. Preferably, the biosynthetic pathway is a polyhydroxyalkanoate biosynthesis pathway.
This disclosure describes multigene vectors designed to produce polyhydroxyalkanoate (PHA) in plants. Some of these vectors are designed to produce poly(xcex2-hydroxybutyrate), and some are designed to produce poly(xcex2-hydroxybutyrate-co-xcex2-hydroxyvalerate) (Gruys et al., WO 98/00557, 1998). In general, the efficiency of PHA production was dramatically increased when all sequences necessary for a pathway were introduced on the same vector. Herein, construction of these multigene vectors, and their use for polyhydroxyalkanoate production in Arabidopsis thaliana and Brassica napus, and Zea mays is described.
An embodiment of the present invention is an isolated nucleic acid segment comprising multiple nucleic acid sequences, each encoding a different protein within the biosynthetic pathway. Preferably, the isolated nucleic acid segment comprises a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; and a third nucleic acid sequence encoding a xcex2-ketothiolase protein. The nucleic acid segment may further comprise additional nucleic acid sequences encoding additional proteins such as a threonine deaminase protein or a deregulated threonine deaminase protein.
An alternative embodiment of the invention is a recombinant vector comprising multiple nucleic acid sequences, each encoding a different protein within the biosynthetic pathway. The recombinant vector may be arranged with a single promoter producing a polycistronic RNA transcript from the multiple nucleic acid sequences, or with each nucleic acid sequence being under the control of its own promoter. The multiple promoters may be the same or different. It is also possible to have one or more nucleic acid sequence under the control of its own promoter, while other nucleic acid sequences may be jointly under the control of a single promoter producing a polycistronic RNA transcript.
A recombinant vector placing the biosynthetic pathway nucleic acid sequences under the control of a single promoter preferably comprises operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a promoter that directs transcription of the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3xe2x80x2 transcription terminator; and a 3xe2x80x2 polyadenylation signal sequence; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; and the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a xcex2-ketoacyl reductase protein, and a nucleic acid sequence encoding a xcex2-ketothiolase protein. The nucleic acid sequences encoding the biosynthetic pathway enzymes may be in any order relative to each other and the promoter. The promoter must be expressed in plastids. It may have either been derived from a plastid, or may have been derived from a bacterium or phage having promoters recognized by the plastid transcription enzymes, or be a synthetic promoter recognized by the plastid transcription enzymes.
A recombinant vector placing the biosynthetic pathway nucleic acid sequences under the control of multiple promoters preferably comprises a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence. The xcex2-ketothiolase protein preferably condenses two molecules of acetyl-CoA to produce acetoacetyl-CoA; and condenses acetyl-CoA and propionyl-CoA to produce xcex2-ketovaleryl-CoA. The xcex2-ketoacyl reductase protein preferably reduces acetoacetyl-CoA to xcex2-hydroxybutyryl-CoA; and reduces xcex2-ketovaleryl-CoA to xcex2-hydroxyvaleryl-CoA. The polyhydroxyalkanoate synthase protein is preferably selected from the group consisting of: a polyhydroxyalkanoate synthase protein that incorporates xcex2-hydroxybutyryl-CoA into P(3HB) polymer; and a polyhydroxyalkanoate synthase protein that incorporates a xcex2-hydroxybutyryl-CoA and a xcex2-hydroxyvaleryl-CoA into P(3HB-co-3HV) copolymer. The xcex2-ketothiolase protein may comprise a transit peptide sequence that directs transport of the xcex2-ketothiolase protein to the plastid. The xcex2-ketoacyl reductase protein may comprise a transit peptide sequence that directs transport of the xcex2-ketoacyl reductase protein to the plastid. The polyhydroxyalkanoate synthase protein may comprise a transit peptide sequence that directs transport of the polyhydroxyalkanoate synthase protein to the plastid. The recombinant vector may further comprise a nucleic acid sequence encoding a threonine deaminase protein or a deregulated threonine deaminase protein. The first promoter, second promoter, and third promoter are preferably active in plants. The first promoter, second promoter, and third promoter are preferably viral promoters. The first promoter, second promoter, and third promoter are preferably independently selected from the group consisting of a CMV 35S promoter, an enhanced CMV 35S promoter, maize chlorophyll A/B binding protein promoter, and an FMV 35S promoter. More preferably, the first promoter, second promoter, and third promoter are the CMV 35S promoter. The first promoter: second promoter, and third promoter may be tissue specific promoters. The first promoter, second promoter, and third promoter may independently be the Lesquerella hydroxylase promoter or the 7S conglycinin promoter, and preferably each is the Lesquerella hydroxylase promoter.
An alternative embodiment is directed towards transformed host cells. Transformed host cells may contain a non-integrated recombinant vector or an integrated recombinant vector.
A transformed host cell may comprise a recombinant vector, wherein the recombinant vector comprises a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence.
The transformed host cell may alternatively contain an integrated nucleic acid segment. Preferably, the transformed host cell may comprise a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence. The first element, second element, and third element may be cointegrated within a continuous 10 Mb segment of genomic DNA, more preferably within a continuous 5 Mb, 2.5 Mb, 2 Mb, 1.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment of genomic DNA. Alternatively, the first element, second element, and third element may be cointegrated between a left Ti border sequence and a right Ti border sequence. While it is preferable that a recombinant vector contain a single left Ti border sequence and a single right Ti border sequence, the invention encompasses recombinant vectors containing multiple left and/or right Ti border sequences, and the use thereof FIG. 2C.
Alternatively, the host cell may comprise a nucleic acid segment containing nucleic acid sequences encoding enzymes in a biosynthetic pathway, where a single promoter directs transcription of the nucleic acid sequences.
The transformed host cell may generally be any host cell, and preferably is a bacterial, fungal, or plant cell. The bacterial cell is preferably an Escherichia coli cell. The fungal cell is preferably a yeast, Saccharomyces cerevisiae, or Schizosaccharomyces pombe cell. The plant cell may be a monocot plant cell, a dicot plant cell, an algae cell, or a conifer plant cell. The plant cell is preferably a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, sugar beet, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
The promoters may be any of the promoters discussed earlier. The transformed host cells preferably produce polyhydroxyalkanoate polymer.
The invention also encompasses transformed plants. The transformed plant may contain an integrated set of nucleic acid sequences, or may contain the same set of nucleic acid sequences on a non-integrated vector. A preferred embodiment is directed towards a transformed plant comprising a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence. The first element, second element, and third element may be cointegrated within a continuous 10 Mb segment of genomic DNA, more preferably within a continuous 5 Mb, 2.5 Mb, 2 Mb, 1.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment of genomic DNA. Alternatively, the first element, second element, and third element may be cointegrated between a left Ti border sequence and a right Ti border sequence FIG. 2C.
Alternatively, the transformed plant may comprise a nucleic acid segment containing nucleic acid sequences encoding enzymes in a biosynthetic pathway, where a single promoter directs transcription of the nucleic acid sequences.
The transformed plant may generally be any type of plant, and preferably is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
The promoters may be any of the promoters discussed earlier. The transformed plant preferably produces polyhydroxyalkanoate polymer.
The invention also encompasses methods of preparing transformed host cells. The methods may produce a transformed host cell having nucleic acid sequences under the control of multiple promoters or under the control of a single promoter. The method preferably comprises the steps of selecting a host cell; transforming the selected host cell with a recombinant vector comprising: a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence; and obtaining transformed host cells; wherein the transformed host cells produce polyhydroxyalkanoate polymer.
Alternatively, the method of preparing transformed host cells may comprise the steps of selecting a host cell: transforming the selected host cell with a recombinant vector comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3xe2x80x2 transcription terminator; and a 3xe2x80x2 polyadenylation signal sequence; and obtaining transformed host cells; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a xcex2-ketoacyl reductase protein, and a nucleic acid sequence encoding a xcex2-ketothiolase protein; and the transformed host cells produce polyhydroxyalkanoate polymer.
The promoters may be any of the promoters discussed earlier.
Also disclosed are methods for preparing transformed plants. The methods may produce a transformed plant having nucleic acid sequences under the control of multiple promoters or under the control of a single promoter. The method preferably comprises the steps of selecting a host plant cell; transforming the selected host plant cell with a recombinant vector comprising: a first element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3xe2x80x2 transcription terminator; and a first 3xe2x80x2 polyadenylation signal sequence; a second element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a xcex2-ketoacyl reductase protein; a second 3xe2x80x2 transcription terminator; and a second 3xe2x80x2 polyadenylation signal sequence; and a third element comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a xcex2-ketothiolase protein; a third 3xe2x80x2 transcription terminator; and a third 3xe2x80x2 polyadenylation signal sequence; obtaining transformed host plant cells; and regenerating the transformed host plant cells to produce transformed plants, wherein the transformed plants produce polyhydroxyalkanoate polymer.
Alternatively, the method of preparing a transformed plant may comprise the steps of selecting a host plant cell; transforming the selected host plant cell with a recombinant vector comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction: a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3xe2x80x2 transcription terminator; and a 3xe2x80x2 polyadenylation signal sequence; obtaining transformed host plant cells; and regenerating the transformed host plant cells to produce transformed plants; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a xcex2-ketoacyl reductase protein, and a nucleic acid sequence encoding a xcex2-ketothiolase protein; and the transformed plants produce polyhydroxyalkanoate polymer.
The promoters may be any of the promoters discussed earlier.
The invention is also directed towards methods of producing biomolecules of interest. The imultiple enzymes in the biosynthetic pathway may lead to the production of materials of commercial and scientific interest. Preferably, the biomolecules are polymers, and more preferably are polyhydroxyalkanoate polymers. The methods may comprise obtaining any of the above described transformed host cells or transformed plants, culturing or growing the transformed host cells or transformed plants under conditions suitable for the production of polyhydroxyalkanoate polymer, and recovering polyhydroxyalkanoate polymer. The methods may further comprise the addition of nutrients, substrates, or other chemical additives to the growth media or soil to facilitate production of polyhydroxyalkanoate polymer. In a preferred embodiment, it is possible to extract the polyhydroxyalkanoate from the transformed host cells or transformed plants without killing the host cells or plants. This may be accomplished, for example, by various solvent extraction methods or by engineering the host cells or plants to secrete the polyhydroxyalkanoate polymer, or by directing production to tissues such as leaves or seeds which may be removed without causing serious injury to the plant. The polyhydroxyalkanoate polymer produced is preferably poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(4-hydroxybutyrate), or poly(3-hydroxybutyrate-co-4-hydroxybutyrate).
If repetitive sequences are used in a multi-gene plasmid system, there exists the possibility for gene silencing in subsequent generations of plants. If expression levels are high gene silencing could also occur and would be independent of repetitive elements. Repetitive sequences may include the use of the same promoters, chloroplast peptide encoding sequences, and other genetic elements for each of the multi-gene coding sequences. Gene silencing often manifests itself as a gradual reduction in protein levels, mRNA levels, or biosynthesis product concentrations in subsequent generations of related plants. If gene silencing is observed, changing the repetitive sequences through the use of diverse genetic elements such as different promoters, leaders, introns, transit peptide sequences, etc., different designed nucleotide sequence, or through mutagenesis of the existing sequence, may be successful in reducing or eliminating the gene silencing effects.